Progestins are a class of naturally occurring steroidal hormones which are produced in the ovaries and other tissues in the body and which directly influence the growth and function of specific target tissues in humans and animals. Although a variety of naturally occurring and chemically synthesized progestins have been identified and characterized (including 17a-hydroxyprogesterone, norethisterone acetate, medroxyprogesterone, and megestrol acetate), perhaps the best known is progesterone [Pregn-4-ene-3,20-dione] which is endogenously secreted during the latter half of the menstrual cycle.
progestins mediate their action by binding to an intracellular protein, presumably a nuclear protein, identified as "progesterone receptor" and also abbreviated as "PR". The presence of this intracellular PR provides and accounts for new protein synthesis by progesterone dependent cells. The progesterone receptor in the absence of the progesterone hormone is biologically inactive both in vivo and in vitro; and, if the cells or tissues are homogenized and fractionated into cytosol and nuclear fractions, the progesterone receptor is found as a soluble protein in the cytosol. Although the precise molecular mechanism of progesterone action remains poorly understood, the generally accepted sequence of events is believed to be as follows: When progesterone is introduced to the target cells and tissues, there is specific binding of progesterone to PR protein which results in the formation of a progesterone/receptor protein complex. Also, at a time subsequent to progesterone binding, a process termed activation and/or transformation ensues which leads to the formation of functional progesterone/receptor complexes having a high affinity for the nuclear components, the DNA, of the target cell. Once the hormone/receptor protein complex is physically formed, it is said to translocate as a complex into the nucleus of the cell where it binds to specific DNA sequences known as progesterone responsive elements on the chromosomes and initiates messenger ribonucleic acid (mRNA) transcription. New messenger RNA is then synthesized, chemically modified, and exported from the nucleus into the cytoplasm of the cell where ribosomes translate the mRNA into new proteins. This is the well-recognized progesterone effect on the cell--that is, the initiation of new protein synthesis.
The theoretical premise and the generally accepted, though poorly understood, mechanism of action regarding progesterone hormones, progesterone receptor proteins, and their interactions are described in greater detail by the following publications--all of Which are merely representative of the ongoing investigations in this field. These are: Horowitz et al., Recent Progress In Hormonal Research 41:249-316 (1985) and the references cited therein; Gronemeyer et al., EMBO J. 6:3985-3994 (1987); Traish et al., Endocrinology 118:1327-1333 (1986); Traish et al, Steroids 47:157-173 (1986); Loosfelt et al , Proc. Soc. Natl. Acad. Sci. USA 83:9045-9049 (1986); and Misrahi et al., Biochem. Biophys. Res. Comm. 143:740-748 (1987); Beato, M., Cell 56:335-344 (1989); Evans, R. M., Science 240:889-895 (1989); Conneely et al., Science 233:767-770 (1986); NaKao, M. and V. K. Moudsit, B.B.R.C. 164:295-303 (1989).
In order to truly appreciate the background of the present invention, it is useful to summarize in depth the major details and sequential events presently believed to occur regarding the intracellular protein referred to as "progesterone receptor." In the unbound state, and in the absence of progesterone hormone, the progesterone receptor protein thought to be located in vitro within the cytosol; however some new evidence suggests that the receptor may be located in the nucleus of the cell. The intact PR protein is composed of 933 amino acids. The native human PR proteins found in soluble tissue extract exist as large molecular weight complexes (250,000-300,000 Da). The physico-chemical properties of these receptor proteins, isolated from various human and non-human sources, have been the subject of intensive investigations. It has been demonstrated that PR complex is almost always composed of two proteins termed A and B subunits with molecular weights of 84 and 110 kDa, respectively. In the untransformed state it is thought that these complexes are associated with heat-shock proteins. The biological significance of these large untransformed complexes in vitro remains as yet unclear.
In cell free systems, the solubilized protein prepared in low salt buffers containing both sodium molybdate and proteolysis inhibitors, the progesterone receptor remains untransformed. The human PR protein can be found in alternative molecular forms which sediment at either 8-10S or 4S as determined by sucrose density gradient analysis. The 8S form of PR is believed to be the unactivated, untransformed polymeric form of the PR protein associated with the unbound, native state of receptor in the absence of progesterone. The 8S PR form is a large molecular weight complex, presumably associated with heatshock proteins, that does not bind to nuclei or DNA in vitro and is stabilized as a macromolecule by sodium molybdate.
In comparison, the 4S PR protein form is a monomer--i.e., the protein molecule that can be generated from the 8S form in vitro by treatment with high ionic strength buffers, by elevated temperatures (25.degree.-30.degree. C.), or by increasing salt concentrations (KCl or NaCl). The 4S PR form functionally binds to both nuclei and DNA-cellulose in vitro; it is generally termed the "activated and/or transformed" progesterone receptor protein. From the published reports, it appears that the dissociation of the 8S PR form into the 4S PR form initiates a major structural change in the stereochemical conformation of the protein or by directly exposing the previously hidden DNA binding domain of the receptor molecule.
It is essential also to recognize that progesterone receptor proteins from human and animal sources have been investigated and evaluated in terms of functional domains which provide and are responsible for its characteristic biological and physiological properties. Complimentary DNA (cDNA) of human progesterone receptor has been cloned which, in turn, has led directly to the elucidation of the human ER protein primary sequence. Subsequent studies have further defined the various functional domains of human PR protein as having at least three distinctly different regions, each of which comprises at least one domain and provides unique functional properties and characteristics. See for example Beato, M., Cell 5:335-344 (1989).
FIG. 1 illustrates the various domains within the PR protein. The amino-terminal "A/B" region is the hypervariable region and is thought to contribute to full functional activity of the receptor. The middle cysteine rich "C" domain is a critical region for biological activity because it is the DNA-binding region required for interaction with progesterone responsive elements ("PRE"). The "D" region is the hinge region; its function is not yet clear. Region "E" is the carboxy-terminal region and is believed to serve as the progesterone binding domain. The "E" region comprises an amino acid sequence which is generally shared not only among various human and non-human sources of PR; but also is conserved among all the different steroid receptor members throughout the different classes--thereby indicating that substantially similar carboxy-terminal "E" domains are critical for receptors generally throughout all the steroid receptors as a family. Specific publications describing these functional domain investigations, data, and conclusions in greater detail are represented by the following: Green et al., Nature 328:134-139 (1986); Hollenberg et al., Nature 318:635-641 ( 1985); Arriza et al., Science 237.:268-275 (1987); Mishraki et al., Biochem. Biophys. Res. Comm. 143:740-748 (1987); Lubahn et al., Science 240:327-330 (1988); Chang et al., Science 240:324-326 (1988); and Beato, M., Cell 56:335-344 (1989).
Overall, it will be noted and appreciated that many investigations of PR protein and the characterization of hormone/PR complexes typically involve immunological methods and assay. A variety of different polyclonal antisera have been prepared against native and fractionated progesterone receptor protein; and against the nuclear binding progesterone receptor complex [Tuohimaa et al., Biochem. Biophys. Res. Comm. 119:433 (1984); Renoir et al., Eur. J. Biochem. 324:1 (1982); Gronemeyer et al., J. Biol. Chem. 260:6916 (1985); Smith et al., Endocrinology 122:2816-2825 (1988); Smith et al., J. Steroid. Biochem. 30:1-7 (1988); Welgel et al., Endocrinology 125:2494-2501 (1989)]. Similarly, some monoclonal antibodies against human and animal progesterone receptor proteins and complexes have been prepared for many different investigational purposes with markedly different degrees of success [Loosfelt et al., Proc. Soc. Natl. Acad. Sci. USA 83:9045-9049 (1986); Sullivan et al., Endocrinology 119:1549-1557 (1986); Clark et al., Endocrinology 121:1123-1132 (1987); Nakoa et al., Can. J. Biochem. Cell Biol. 63:33-40 (1985); Hendler and Yuan, Cancer Research 45:421-429 (1985); and U.S. Pat. No. 4,742,000].
The common drawback and recurring problem of these known polyclonal and monoclonal antibodies is their uniform and consistent failure to be site-specific or site-selective for a defined domain of PR. This failure, in turn, produces erroneous empirical results and unreliable information- not only for investigational purposes but also in clinical applications of such antibodies for therapeutic purposes. As a major example, the measurement of progesterone receptors in human breast carcinomas has been one aspect of the primary tool and favored diagnostic method (in addition to the measurement of estrogen receptor) for choosing between hormonal and cytotoxic chemotherapy when treating breast cancer patients. A commercially prepared immunoassay kit employing anti-PR antibodies is most commonly used for this purpose [The Abbott PgR-ICA Monoclonal Immunocytochemical Assay; Greene, G. and M. F. Press, Immunological Approaches To The Diagnosis And Therapy of Breast Cancer (R. Ceriani, editor) , Plenum Publishing Co., New York, 1987, pp 119-135]. Unfortunately, this immunoassay kit employs conventionally obtained monoclonal antibodies prepared against purified whole PR protein for these measurements. These monoclonal antibodies have been found to be frequently unreliable and often insufficiently specific. Clearly, therefore, given all the presently known antibodies, assays, and immunological techniques, one still cannot accurately predict which of these progesterone receptor positive tumors will respond to hormonal treatment.
The causes of the present dilemma are in fact two fold: First is the failure of the known monoclonal antibodies and polyclonal antisera to be sufficiently domain-specific and epitope-selective in order to demonstrate the presence of unfragmented progesterone receptor. Second is the failure (insofar as is presently known) of conventional antibodies to be able to identify the true functional status of progesterone receptor protein using an immunoassay system. It is now clearly apparent to practitioners and clinicians ordinarily skilled in this art that so long as these insufficiently-specific antibodies remain in clinical use, many repetitive failures of the known immunoassay systems will occur; and the clinician's ability to identify that proportion of breast cancer patients which would be sensitive and responsive to hormonal treatment will remain plagued with uncertainty and inaccuracy. For these reasons, the development of domain-specific antibodies which could then be employed within conventional diagnostic immunoassays would therefore be recognized generally as a major advance and fundamental improvement in antibody materials, assay reliability, and therapeutic benefit.